NMR-based Metabolomics Analysis*

Eurycoma longifolia (Tongkat Ali, TA), native to Malaysia and South-East Asia, have been traditionally used as a remedy to boost male sexual desire, libido, energy and fertility.  Among the different types of extract, water extract is the most common type of TA extract.

Male infertility is one of the leading causes of infertility which affects many couples worldwide. Semen analysis is a routine examination of male fertility status which is usually performed on semen samples obtained through masturbation. Sperm count increased, a quantitative measurement to fertility, could be associated with metabolic changes in system biology. Application of metabonomics approaches on animal fertility study could lead to the discovery of potential biomarkers.

The study was first carried out to look for metabolic discrimination among TA roots from different geographical terrains, especially with respect to the biologically active quassinoids¹. The analysis results might be useful in that it could suggest the geographical location containing TA plants rich in quassinoids. Quassinoids are the indigenous metabolites of the plants from the Simaroubaceae family and have been proven to be responsible for many of the therapeutic properties of the plant, especially the enhancement of male fertility.

Subsequently, animal study was carried out to evaluate the effect of TA extracts with different quassinoid levels on rats sperm count and examined corresponding post-treatment urinary metabolic changes2. The study was also aimed at investigating potential plasma biomarkers associated with the sperm count increase in rats³. As semen samples obtained through masturbation may be inconvenient to patients, this approach may be considered as a path towards a supplement/ alternative to the evaluation of male fertility status in humans.

a) Plant Study¹
TA root aqueous extracts from Perak (n = 30), Selangor, Kedah, Terengganu (n = 5 for each) were checked for metabolic discrimination via a combined 1H-NMR spectroscopy and Orthogonal Partial Least-Squares Discriminant Analysis (OPLS-DA) as chemometric data analysis technique. Chemometric model enabled a clear separation of the samples based on their origin and, consequently, choline, lactic acid, succinic acid, eurycomanol, and eurycomanol-2-O--D-glucopyranoside were detected as the potential discriminatory metabolites. Terengganu and Perak samples contained higher amounts of eurycomanol and eurycomanol-2-O-β-D-glycopyranoside, respectively.

Subsequently, 1H‑NMR spectra of TA root aqueous extracts from Perak were used as plant reference standard for identification of primary metabolites and the quassinoids. In addition, quantitative NMR approach was also employed towards measuring quassinoid levels in TA roots from different states in Malaysia.

b) Animal Study^2,3
24 male Sprague–Dawley rats, categorized into 4 groups of 6 rats each, were orally administered for 48-days with water for the control (Group 1), TA water extract (Group 2), TA quassinoid-poor extract (Group 3) and TA quassinoid-rich extract (Group 4). Upon completion of the 48-day treatment, the animals were subsequently sacrificed for sperm count analysis. Post-treatment urine and plasma were collected and analyzed by NMR. The urine and plasma profiles were categorized according to sperm count level.

The results showed that the sperm count in Group 2 and Group 4 were significantly higher compared to the Group 1 and Group 3. The OPLS-DA model indicated a clear separation among the urine profiles with respect to sperm count level. Consequently, the rats were divided into normal- (Group 1 and Group 3) and high- (Group 2 and Group 4) sperm count groups. 1H-NMR profiles of the high-sperm count group contained higher concentrations of trigonelline, alanine, benzoic acid and higher intensity of a signal at 3.42 ppm, while ethanol was at higher concentration in the normal-sperm count group. Therefore, the results proved the efficacy of quassinoids on sperm count increase in rats and provided quantitative markers in urine suitable for analysis of sperm profile and male fertility status.

Carr-Purcell-Meibum-Gill (CPMG) experiments combined with OPLS-DA was employed to evaluate plasma metabolomic changes in normal and high sperm count rats. The potential biomarkers associated with sperm count increase were investigated to assess fertility by capturing the metabolomic profile of plasma. The results indicated that high sperm count groups contained higher concentrations of alanine, lactate, and histidine in relation to the total plasma profile. However, the plasma concentration of ethanol was higher in the normal sperm count group as compared to the high sperm count group.

References:

1. Forough Ebrahimi, Baharudin Ibrahim, Chin-Hoe Teh, Vikneswaran Murugaiyah & Kit-Lam Chan (2017). NMR-based plasma metabolomic discrimination for male fertility assessment of rats treated with Eurycoma longifolia extracts. Systems Biology in Reproductive Medicine 63 (3) 179–191.
2. Forough Ebrahimi, Baharudin Ibrahim, Chin-Hoe Teh, Vikneswaran Murugaiyah and Kit-Lam Chan (2017). 1H NMR-based discriminatory analysis of Eurycoma longifolia from different locations and establishing a profile for primary metabolites identification and quassinoids quantification. Planta Medica, 83:172-182.
3. Forough Ebrahimi, Baharudin Ibrahim, Chin-Hoe Teh, Vikneswaran Murugaiyah and Kit-Lam Chan (2016). Urinary NMR-based metabolomics analysis of rats possessing variable sperm count following orally administered Eurycoma longifolia extracts of different quassinoid levels. Journal of Ethnopharmacology, 182, 80-89.

Written by Chin-Hoe The, Bruker.  Chin-Hoe obtained his PhD in Pharmaceutical Chemistry from University of Sains Malaysia. He joined Bruker Malaysia as a NMR Application Scientist and supports the South East Asia region and provides the assistance to the customers especially who are new in the natural products – plant metabolomics fields.